Lab#1 Microscopy & Epithelial Tissue Lama AlAbdi Supervised by: Dr. Reem AlAjmi
Rapid examination of fresh tissue using light-sheet microscopy€¦ · Rapid examination of fresh...
Transcript of Rapid examination of fresh tissue using light-sheet microscopy€¦ · Rapid examination of fresh...
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Rapid examination of fresh tissue using light-sheet microscopy
Nicholas P. Reder, MD, MPH 11/7/2017
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Nicholas P. Reder, MD, MPH
• Earned a B.S. from the
University of Michigan
• Earned an M.P.H. in
epidemiology from Emory
University
• Earned MD from Loyola
Stritch School of Medicine in
2014
• Genitourinary pathology
fellow in the University of
Washington Department of
Pathology.
© 2017 College of American Pathologists. All rights reserved.
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Disclaimer
• The CAP does not permit reproduction of any substantial
portion of the material in this Webinar without its written
authorization. The CAP hereby authorizes attendees of the
CAP Webinar to use the PDF presentation solely for
educational purposes within their own institutions. The CAP
prohibits use of the material in the Webinar – and any
unauthorized use of the CAP’s name or logo – in connection
with promotional efforts by marketers of laboratory
equipment, reagents, materials, or services.
© 2017 College of American Pathologists. All rights reserved.
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Disclaimer
• Opinions expressed by the speaker are the speaker’s own
and do not necessarily reflect an endorsement by the CAP of
any organizations, equipment, reagents, materials, or
services used by participating laboratories.
© 2017 College of American Pathologists. All rights reserved.
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Disclosure
• Dr. Reder holds a patent and has a start-up
company (Alpenglow Optics, LLC) related to light-
sheet microscopy work.
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Learning Objectives
• Understand the different techniques for
examination of fresh tissue
• Be able to articulate the strengths and weaknesses
of each technique
• Describe use-cases for slide-free microscopy of
fresh tissue
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Outline
• Motivation for slide-free microscopy of fresh tissue
• Overview of slide-free microscopy techniques
• False-coloring to mimic H&E
• Use-cases
• Summary
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Outline
• Motivation for slide-free microscopy of fresh tissue
• Overview of slide-free microscopy techniques
• False-coloring to mimic H&E
• Use-cases
• Summary
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4. Section
~10 minutes
3. Embed
~10 minutes
2. Process
~2.5 hours
1. Fix
~30 minutes
5. Stain
~40 minutes
Histology
Histology
or
1. Freeze
~2 minutes
2. Section
~2 minutes
3. Stain
~4 minutes
Rapid histology: 4 hours Frozen Section: 10 minutes
Motivation: pathology has remained unchanged for a century
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4. Section
~10 minutes
3. Embed
~10 minutes
2. Process
~2.5 hours
1. Fix
~30 minutes
5. Stain
~40 minutes
Histology Expensive
Time-consuming
Destructive
Hazardous
Sampling errors
Disadvantages
Histology
or
1. Freeze
~2 minutes
2. Section
~2 minutes
3. Stain
~4 minutes
Rapid histology: 4 hours Frozen Section: 10 minutes
Destructive
Hazardous
Sampling errors
Variable histology quality
Disadvantages
Motivation: pathology has remained unchanged for a century
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2. Image
<10 minutes
1. Stain
<1 minute
Wide-area ‘digital’ histology
Fast
Digital
Non-destructive
Slide-free
Wide-area
Advantages
Goal: non-destructive, slide-free, ‘digital’ pathology
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Outline
• Motivation for slide-free microscopy of fresh tissue
• Overview of slide-free microscopy techniques
• False-coloring to mimic H&E
• Use-cases
• Summary
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Contrast / depth
Speed Resolution
Low cost Ease-of-use
Surface imaging
Confocal, Nonlinear MUSE Structured-illumination Light-sheet
Microscopy method
Overall comparison of fluorescence
microscopy technologies
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Similarities across all techniques
• Slide-free imaging Efficient workflow
• Non-destructive Preservation of tissue for molecular testing
• Fluorescence imaging: need to use topically applied fluorescent dyes,
or endogenous fluorophores (autofluorescence)
• Other non-fluorescent techniques can achieve slide-free, non-
destructive imaging, but they are beyond the scope of this presentation
• OCT
• Photoacoustic
• Spectroscopy
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MUSE
• Microscopy with UV Surface Excitation
• UV light has limited penetration (10 microns)
• Advantages
• Fast
• Simple
• Inexpensive
• High resolution
• Disadvantages
• Surface imaging only
Fereidouni F, Harmany Z, Demos S, Levenson R. MUSE: Microscopy via UV excitation for
rapid histology. In: Photonics Conference (IPC). 2016 IEEE 2016 Oct 2 (pp. 146-147). IEEE.
XYZ
stage
LED
Objective
Tube lens
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MUSE
How does MUSE produce images?
Limited penetration of UV light “Optical section”
Figure courtesy of Dr. Yu “Winston” Wang, University of Washington, Department of
Mechanical Engineering
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Single-excitation
source, color CCD
camera
Orange: elastic
laminae in artery
Blue: collagen
Green, orange
and light blue:
renal tubules and
nuclei
Slide courtesy of Dr. Richard Levenson, UC Davis
MUSE
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Slide courtesy of Dr. Richard Levenson, UC Davis
SEROMUCINOUS OVARIAN CARCINOMA
MUSE
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Slide courtesy of Dr. Richard Levenson, UC Davis
MUSE
• Example: Skin
• Easy distinction
between elastin
(yellow) and
collagen (green)
• Usually requires
special stains
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Structured Illumination
• Structured illumination
microscopy
• Uses patterned light to
improve resolution
• Advantages
• Fast
• High resolution
• Disadvantages
• Modest imaging depth
• Complex optics
Fu HL, Mueller JL, Javid MP, Mito JK, Kirsch DG, Ramanujam N, Brown JQ. Optimization
of a widefield structured illumination microscope for non-destructive assessment and
quantification of nuclear features in tumor margins of a primary mouse model of sarcoma.
PloS one. 2013;8(7):e68868.
Uniform illumination Structured illumination
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Structured Illumination
Wang M, Kimbrell HZ, Sholl AB, Tulman DB, Elfer KN, Schlichenmeyer TC, Lee BR, Lacey M,
Brown JQ. High-resolution rapid diagnostic imaging of whole prostate biopsies using video-
rate fluorescence structured illumination microscopy. Cancer research. 2015;75(19):4032-41.
Example: Prostate
Easy distinction of
benign and
neoplastic lesions
High accuracy:
AUC of 0.82-0.88
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Confocal
• Confocal microscopy
• Uses a pin-hole to reject out-
of-focus light
• Advantages
• Depth of imaging
• High resolution
• Commercially available
• Disadvantages
• Requires elaborate tissue
flattening for surface imaging
• Slow (in 3D) due to point-scanning
Gareau DS, Patel YG, Li Y, Aranda I, Halpern AC, Nehal KS,
Rajadhyaksha M. Confocal mosaicing microscopy in skin
excisions: a demonstration of rapid surgical pathology. Journal of
microscopy. 2009;233(1):149-59.
Epifluorescence Confocal
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Confocal
Ragazzi M, Piana S, Longo C, Castagnetti F, Foroni M, Ferrari G, Gardini G, Pellacani
G. Fluorescence confocal microscopy for pathologists. Modern Pathology.
2014;27(3):460-71.
Example: Thyroid
Easy distinction of
benign and
neoplastic lesions
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Multiphoton
• Multiphoton microscopy
• Aka two-photon, nonlinear
• Uses a pulsed laser to
achieve precise localization
of excitation
• Advantages
• Depth of imaging
• Very high resolution
• Disadvantages
• Requires elaborate tissue
flattening for surface imaging
• Slow (in 3D) due to point-scanning
• Expensive and complex optics
Yoshitake T, Giacomelli MG, Cahill LC, Schmolze DB, Vardeh H,
Faulkner-Jones BE, Connolly JL, Fujimoto JG. Direct comparison
between confocal and multiphoton microscopy for rapid
histopathological evaluation of unfixed human breast tissue.
Journal of Biomedical Optics. 2016;21(12):126021-.
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Multiphoton
Images: Yoshitake T, Giacomelli MG, Cahill LC, Schmolze DB, Vardeh H, Faulkner-Jones BE, Connolly JL, Fujimoto JG.
Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human
breast tissue. Journal of biomedical optics. 2016;21(12):126021-.
1. Tao YK, Shen D, Sheikine Y, Ahsen OO, Wang HH, Schmolze DB, Johnson NB, Brooker JS, Cable AE, Connolly JL,
Fujimoto JG. Assessment of breast pathologies using nonlinear microscopy. Proceedings of the National Academy of
Sciences. 2014;111(43):15304-9.
Example: Breast, invasive ductal carcinoma
Crisp nuclear detail, easy diagnosis (>94% accuracy)1
Multiphoton Confocal H&E
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Advantage: LSM rapidly images a 3D volume, within which an irregular
tissue surface may be digitally extracted and imaged over a range of
depths
Shallow
depth
of focus
Deep
depth
of focus
Conventional microscopy Light-sheet microscopy
Irregular tissue surface
100 μm
Tissue
irregularit
y
LSM for imaging fresh tissues
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LSM Demonstration
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Outline
• Motivation for slide-free microscopy of fresh tissue
• Overview of slide-free microscopy techniques
• False-coloring to mimic H&E
• Use-cases
• Summary
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DRAQ5
Eosin
Merged
DRAQ5 and Eosin dual-channel fluorescent staining and imaging of
human prostate core-needle biopsy
1 mm
False-colored
1 mm
False-color H&E imaging
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Nuclear stain (DRAQ5, 𝜆𝑒𝑥 = 660 nm, 𝜆𝑒𝑚 = 680 nm)
Cytoplasmic stain (Eosin, 𝜆𝑒𝑥 = 488 nm, 𝜆𝑒𝑚 = 500 nm)
‘Digital’ histology
False-color H&E imaging
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MUSE H&E
False-color H&E imaging
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H&E GUI – Tune the stain to your liking
Slide courtesy of Dr. Richard Levenson, UC Davis
MUSE
Kidney
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Outline
• Motivation for slide-free microscopy of fresh tissue
• Overview of slide-free microscopy techniques
• False-coloring to mimic H&E
• Use-cases
• Summary
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Radical prostatectomy
Prostate
slices
(3-5 mm
thick)
1 cm
Use-case: Post-operative triaging of fresh tissue
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1 cm
Fresh prostate
slice
Excis
ed
pro
sta
te
1 cm
Prostate slices
(3-5 mm thick)
Acridine
Orange
Staining time: 20 sec.
Imaging time: ~8 min
Tissue size: ~3.4x3.6 cm
Resolution: 1.25
μm/pixel
Use-case: Post-operative triaging of fresh tissue
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2 mm
Light-sheet microscopy of prostate tissue
After surface
extraction
Before surface
extraction
25
0 µ
m
Tilt, ~2 µm/mm (slope =
0.2%)
Tissue surface profile (irregularities, <200
µm)
80 µm
Normal prostate glands
100 µm
Prostate adenocarcinoma
40 µm
80 µm
30 m 30 µm
40 µm
15 µm 15 µm
Use-case: Post-operative triaging of fresh tissue
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21
2 3
17
4
5
6 7
8
9
10 11
18
12
13
14 15
20
16
19
23 24 25
22
1
Fresh
prostate
slice
LSM
image
FFPE
H&E
slide
Clinical correlation study
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2 mm After surface
extraction
Before surface
extraction
25
0 µ
m
Tilt, ~2 µm/mm (slope =
0.2%)
Tissue surface profile (irregularities, <200
µm)
24 tissue samples • 12 benign
• 12 carcinoma
Sensitivity: 0.92
Specificity: 0.92
*Detected 2 cases
of positive margins
missed on 2D
section
Clinical correlation study
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Lumpectomy
Breast slice
(3-5 mm thick) 1 cm
H&E
Fresh breast
tissue
5 mm
5 mm
Acridine
Orange
Staining time: 20 sec.
Imaging time: ~5 min
Tissue size: ~2.1x1.8 cm
Resolution: 1.25
μm/pixel
Intra-operative imaging of breast tissue
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Invasive ductal carcinoma with adjacent normal breast
tissue
100 μm
100 μm
20 μm
20 μm
Adipose tissue
150 μm 150 μm
50 μm
150 μm
50 μm
Light-sheet microscopy
of fresh breast tissue Formalin-fixed
paraffin- embedded
section
Frozen tissue
section
50 μm
60 μm 60 μm
Benign breast lobules
100 μm
Intra-operative imaging of breast tissue
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Intra-operative imaging of prostatectomies - SIM
Wang M, Tulman DB, Sholl AB, Kimbrell HZ, Mandava SH, Elfer KN, Luethy S, Maddox MM, Lai W, Lee BR,
Brown JQ. Gigapixel surface imaging of radical prostatectomy specimens for comprehensive detection of
cancer-positive surgical margins using structured illumination microscopy. Scientific reports. 2016;6:27419.
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Intra-operative imaging of prostatectomies - SIM
Wang M, Tulman DB, Sholl AB, Kimbrell HZ, Mandava SH, Elfer KN, Luethy S, Maddox MM, Lai W, Lee BR,
Brown JQ. Gigapixel surface imaging of radical prostatectomy specimens for comprehensive detection of
cancer-positive surgical margins using structured illumination microscopy. Scientific reports. 2016;6:27419.
Imaging of entire
surface of radical
prostatectomy specimen
~60 cm2, ~15 minutes
Identification of key
structures – nerves,
adipose, carcinoma,
benign glands
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Outline
• Motivation for slide-free microscopy of fresh tissue
• Overview of slide-free microscopy techniques
• False-coloring to mimic H&E
• Use-cases
• Summary
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Summary
• Slide-free imaging Efficient workflow
• Non-destructive Preservation of tissue for molecular
testing
• Digital Use digital pathology tools for quantification,
annotation, etc.
• Multiple options (MUSE, SIM, Confocal, MPM, LSM)
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Summary (continued)
• Use-cases
– Triaging of large surgical specimens
– Triaging of small biopsies
• Permanent digital record
• Entire specimen can be sent fresh for molecular studies
– Intraoperative imaging
• Large surface areas
• Fatty tissue
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NIH / NCI - Pacific Northwest Prostate Cancer SPORE P50CA97186
NIH / NIDCR – R01 DE023497
NIH / NCI – R01 CA175391
Department of Defense Prostate Cancer Research Program
NIH / NCI - PO1 CA163227
UW Royalty Research Fund
ITHS Collaboration Innovation Award
UW CoMotion Innovation Award
Gordon and Betty Moore Foundation - Data Science Environments Project Award
Alfred P. Sloan Foundation Award
UW Mech Engin
Dr. Jonathan Liu Lab
Dr. Adam Glaser
Ms. Ye Chen
Dr. Yu “Winston” Wang
Mr. Peter Wei
Mr. Chengbo Yin
UW Pathology
Dr. Nick Reder
Dr. Lawrence True
Ms. Erin McCarty
UW eScience
Dr. Ariel Rokem
Dr. Amanda Tan
Dr. Rob Fatland
UW CoMotion
Forest Bohrer
Ken Myer
Mike Connolly
UC Davis (MUSE)
Dr. Richard Levenson
Acknowledgements
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Upcoming Webinars
Register for upcoming & archived webinars:
www.cap.org > Calendar > Webinars
DATE TOPIC SPEAKER(s) 12/5 Role of Reflectance Confocal
Microscopy in Skin Inflammations
Babar K. Rao, MD
© 2017 College of American Pathologists. All rights reserved.
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• The IVM resource guide highlights current IVM
articles and other resources that assist in
understanding and potentially adopting IVM
and EVM
– Printed guides are available for members
($39) and non-members ($69)
– The digital copies of all four Resource
Guides are a complimentary member
benefit
– Access them www.cap.org > Resources
and Publications
The CAP In Vivo Microscopy Resource
Guide – see handout
© 2017 College of American Pathologists. All rights reserved.
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IVM Short Presentations on Emerging
Concepts (SPECs) – see handout • IVM SPECs are:
– Short PowerPoints, created for pathologists
– Useful for educating pathologists
colleagues about IVM and GI specialist on
the role and value of pathologists in IVM
• IVM SPEC Topics:
– In Vivo Microscopy (IVM): A New Role for
Pathologists
– IVM of the GI Tract
– Ex Vivo Microscopy (EVM): A New Tool for
Pathologists
Access them www.cap.org > Resources
and Publications © 2017 College of American Pathologists. All rights reserved.
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IVM Topic Center Page on CAP.ORG
• Check the IVM Topic Center for continued updates
and for all your IVM resources
www.cap.org > Search for “IVM Topic Center”
© 2017 College of American Pathologists. All rights reserved.
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• Thank you for attending our webinar “Rapid
examination of fresh tissue using light-sheet
microscopy” by Nicholas P. Reder, MD, MPH.
– For comments about this webinar or suggestions
for upcoming webinars, contact [email protected]
– NOTE: There is no CME/CE credit available for
today’s complimentary webinar. The pdf of the presentation
will be sent out in a week.
THANK YOU!
© 2017 College of American Pathologists. All rights reserved.
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